Abstract 3047: Mitochondrial DNA copy variation and TFAM expression in astrocytoma

Abstract

Mitochondrial dysfunction plays a role in determining the phenotype through bioenergetic depletion and increased production of ROS in several diseases, including cancer. We have previously demonstrated a relevant reduction of mitochondrial DNA (mtDNA) copy number in astrocytoma of different grades of malignancy, predominantly in grade IV-glioblastoma (GBM). We observed a stepwise increase of TFAM in parallel to the increase of malignancy, and TFAM expression was higher in GBM patients with overall survival longer than 24 months than less than 12 months. TFAM is codified in the nucleus and transported into mitochondria, where is essential for mtDNA replication, repair and protection by forming nucleoid complexes. To further understand the role of TFAM in astrocytoma progression we performed TFAM immunohistochestry in human astrocytoma samples, confocal analysis in GBM cell lines, in vitro functional assays after silencing TFAM by siRNA in two GBM cell lines (U87MG, A172), and high throughput RNA-Seq in Illumina platform for network analysis by Metacore. TFAM protein was detected in cytoplasm in granular pattern of low grade astrocytomas, and also in nuclei of malignant atsrocytomas. Such localization was also confirmed by confocal analysis in two GBM cell lines. TFAM-siRNA transfection decreased TFAM expression level >80% in D2, which persisted up to D7 with evident decrease of mtDNA copy number after 4 days, and maintained until 7 days. Interestingly, a decrease in migration but not in proliferation was observed in both cell lines after TFAM knockdown. Comparison of RNA-Seq of TFAM-siRNA to NTC- siRNA, with 20 million of reads in each experiment performed in duplicate, resulted in 1000 genes differentially expressed and among them 315 were detected in both cell lines. These genes were in pathways related to cell adhesion (integrin-mediated: p = 5.79e-5, histamine H1 receptor signaling in the interruption of cell barrier integrity: p = 3.98e-5, chemokines-mediated: p = 1.22e-5) and ECM remodeling (p = 9.17e-5), supporting the results of in vitro migration assay. 82 genes were downregulated together with TFAM in both cell lines. These genes were in pathways related to immune response IL15 signaling via JAK-STAT cascade involving MyD88-independent toll-like receptor signaling pathway involving ADAM17 and STAT2 (p = 1.89e-5), regulation of cytoskeleton proteins (p = 1.65e-2), hypoxia-induced EMT in cancer and fibrosis (p = 3.37e-2), polyamine and arginine metabolism (p = 2.73e-2, p = 4.65e-2) and as expected, regulation of apoptotic process (p = 9.50e-21). The role of TFAM in cytoskeleton and ECM remodeling may worth additional studies. Also, TFAM in extracellular space, as released by necrotic cells, may potentiate mitochondrial N-formyl peptide-induced secretion of chemokines and activate inflammatory responses, which may open another strategy to further understand TFAM role in tumorigeniesis. Citation Format: Suely K. Marie, Roseli Silva, Antonio Lerario, Miyuki Uno, Sueli Mieko Oba-Shinjo. Mitochondrial DNA copy variation and TFAM expression in astrocytoma. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 3047. doi:10.1158/1538-7445.AM2015-3047